The aim is to evaluate a novel technology for testing the susceptibility of prokaryotes to certain antibiotics, with the goal of developing rapid antibiotic susceptibility tests for Mycobacterium tuberculosis and other pathogens. The technology will rapidly detect growth of resistant bacteria challenged with antibiotics in pure of mixed cultures, using oligonucleotide probes for two RNA targets: ribosomal RNA precursors (pre-rRNA), and mature ribosomal RNA (rRNA). Cellular copy number of pre-rRNA sequences are very sensitive to rifampicin, declining rapidly upon exposure from high to undetectable levels. By probing for these sequences in cells challenged briefly with rifampicin, it will be possible to detect growing (resistant) cells in a single measurement. Mature rRNA has species-specific sequences useful for detection of bacteria in mixed cultures containing other species. Probes for mature rRNA will be combined with probes for pre-rRNA in a rapid, sensitive, easy-to-use assay that simultaneously identifies bacterial species in primary enrichment cultures, and determines their susceptibility to rifampicin and other antibiotics. Such a test would predict the outcome of antibiotic treatment much more rapidly and accurately than conventional tests. In Phase I of the project, we will test the feasibility of the concept using a fast-growing, well characterized bacterium, Escherichia coli. Experiments will focus initially on resistance to rifampicin, one of the "first-line" antibiotics for treatment of tuberculosis, and then on other antibiotics. In Phase II, we will transfer the technology to the more challenging Mycobacterium tuberculosis system. Ultimately, we will apply the technology to other clinically important microorganisms as well.